Visualizing the segregation of CD4 and CD8 T_M reservoirs in mice. (A) Schematic representation of dual-reporter retrovector MFG. LTR indicates long-term repeats; Fluc, firefly luciferase; P2A, porcine teschovirus 2A sequence; EGFP, enhanced green fluorescence protein; and WRE, woodchuck responsive element. (B,C) Visualization of the CD4 and CD8 T_M formation in albino B6 mice postadoptive transfer of 1 × 10⁶ MFG-labeled OT2 (CD4) or OT1 (CD8) effector T cells (T_Es) using BLI. Representative BLI images (B) and the measurements of total body luminescence (TBL, mean ± SEM; C) are shown (n = 4). Shaded area marks contraction phase (C). (D,E) Visualization of the CD4 and CD8 T_Ms in individual tissues using excised tissue BLI. (D) Tissues were collected from albino B6 recipient mice 2 months after transfer of 10 × 10⁶ MFG-labeled OT2 (CD4) or OT1 (CD8) T_Es. PLN indicates peripheral lymph node; MLN, mesenteric LN; SP, spleen; BM, bone marrow; Thy, thymus; GT, genital tract; and Savi, salivary gland. (E) Detailed analysis of CD4 T_M homing to gut. BLI images of the gut tract (top, the “hot spots” are numbered), identification of the “hot spots” as Peyer patches (PPs; middle), and the measurements of luminescence in gut sub-regions (bottom) are shown. Data are representative of 3 independent experiments. (F) Visualization of the antigen-specific or polyclonal CD4 and CD8 T_Ms formed in albino B6 recipient mice each receiving 1 × 10⁶ either MFG-labeled OT2 or OT1 T_Es, or MFG-labeled B6 CD4 or CD8 T_Es using BLI. Representative BLI images collected 1 month after transfer are shown (n = 4). Schematic showing the individual tissue localization in mice is provided for reference. CLNs indicate cervical LNs; A/BLNs, axillary/brachial LNs; and IgLN, inguinal LNs. PLNs refer to the combination of CLNs, A/BLNs, and IgLNs.